Abstract
The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.
Highlights
In contrast to the catalytic center, the lipid composition of the cytochrome c oxidase (CcO) containing membranes shows considerable variations among different species: Bacteria are often rich in phosphatidylethanolamine (PE) [10,11], while the lipid composition of mitochondrial membranes is typically dominated by phosphatidylcholine (PC) alone or a mixture of PC and PE [12]
CcOs were reconstituted into the membrane of large unilamellar vesicles (LUVs) and the lumen of formed proteoliposomes was filled with the pH sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS; Figure 1a) [30,31]
Changes of the intensity ratio I405/I458 and the correspon3doifn1g7 ensemble-averaged pH values are shown for proteoliposomes formed by reconstituting R. sphaeroides CcO into liposomes made of a crude extract of soybean lipids, which is a lipid composition commonly cuasnedbeincfounnvcetriotendalinsttuodtihees loufmreisnpailrpatHorvyaelnuzeybmaseesd(soene Tcaalbiblera1tifoonr tehxepleipriimd ecnomts,pionsiwtihoincshuHsePdTiSnwthaiss esxtupdoyse) d[3t2o].solutions of known pH values [28]
Summary
In the last steps of this process, membrane-bound enzymes couple electron-transfer reactions to translocation of protons in order to generate and maintain an electrochemical gradient across membranes [1,2,3]. CA is known to interact with high affinity with a large number of proteins present in bacterial or mitochondrial membranes, including cytochrome c and CcO [16,17] These interactions, typically mediated by hydrogen bonds to the phosphate groups of CA and van der Waals interactions to the acyl chains of CA [16,17], make CA an important structural constituent of the respiratory chain [18]. Previous studies using a similar enzyme show an influence of membrane composition on enzymatic activity and proton translocation along the membrane [24,25] Motivated by these considerations, we set out to investigate the influence of the lipid environment on the proton turnover rate of R. sphaeroides CcO. In comparison to the microscopy-based assay, this approach offers a higher signal-to-noise ratio in the recording process but does not permit a direct extraction of the rate of proton turnover
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.